This invention relates to dialysis cells. More particularly, the invention relates to a disposable cell that is economical to manufacture and easy to use.
There has been an effort over the last 10 years to develop for clinical and clinical research laboratories some method of estimating free hormone concentrations other than by measuring them indirectly. Equilibrium dialysis is regarded as the best method of separating protein-bound ligand from free ligand, and is used particularly in the thyroid field where iodinated tracers are used. However, it is considered to be a cumbersome procedure, difficult to do, and entirely outside the purview of routine clinical chemistry.
Two factors have contributed to this. One is that there is a mystique about how to measure the dialyzable fraction of thyroxine, which involves equilibrium dialysis of serum to which a tracer amount of radioiodine labelled thyroxine has been added. That mystique is, in part, due to a less than fully understood de-iodination of thyroxine that occurs immediately after its preparation. If a laboratory were to buy radio-iodine labelled thyroxine from a company that sells radio nuclides, by the time the shipment got to the laboratory, the product would be contaminated with radio-iodides and some radio-iodine labelled thyroxines. There are methods available for repurifying that material in the laboratory just before its use; however, during the incubation (which is necessary to achieve equilibrium), further de-iodination occurs, so that the dialysate radioactivity is always made up of at least two molecular species: radio-iodide, which may have been generated during the dialysis incubation, and the radio-iodine labelled thyroxine. The relationship of the one molecular species to the other varies depending on the clinical state of the patient from whom the serum is taken.
With respect to the dialysis itself, published studies indicate that it is important to hold the chemical composition of the serum during dialysis at a physiologic constant. In an effort to get around this iodide contamination problem, and because iodide is not found in serum proteins, almost all dialysis chemistries previously devised for the measurement of free hormones, including thyroxine, employ simple buffers that radically distort the ionic environment of the serum proteins and dilute the serum proteins. As a result, one cannot obtain an accurate measurement of, for example, the dialyzable free thyroxine fraction using undiluted serum samples, unless a large dialysate volume is used to dilute out the iodide.
Standard methods for the measurement of free thyroxine in serum involve dialysis to separate the free form from the protein-bound form. The partitioning of thyroxine between the free and bound forms is estimated by the addition of radioiodine-labeled thyroxine to the serum sample prior to dialysis. The dialysis is carried out by using a diluted serum sample and/or a great excess of dialysate volume to assist in controlling pH (which has a profound effect on T.sub.4 binding to serum proteins) and to help in minimizing the effect of contaminating iodide which poses a major methodologic difficulty. Direct radioimmunoassays of T.sub.4 in serum dialysates in an effort to avoid the tracer T.sub.4 induced artifact resulting from spontaneous deiodination and radioiodide contamination of tracer T.sub.4 have been described previously.
It has now been discovered that the rate of tracer T.sub.4 deiodination during the equilibrium dialysis incubation is different for different sera and that radioiodide contamination of tracer T.sub.4 is different in the dialysates of different sera. It has also been discovered that the effect of diluting serum proteins is different on sera from different clinical disorders.
It is clear that it would be desirable to measure free T.sub.4 concentrations by a method which distorts the endogenous environment as little as possible. Such a method would employ a direct measurement of free T.sub.4 by radioimmunoassay and avoid the addition of radioiodine-labeled T.sub.4 tracers. Furthermore, it would dilute the serum sample as little as possible, employ a buffer which is as much like an ultrafiltrate of serum as is possible and carry out the dialysis procedure not only at physiologic temperatures but also in an environment of gases which mimic the physiologic in vivo situation.
In one of its embodiments, the dialysis cell of this invention is designed to accomplish this by allowing the dialysis of a small volume of buffer against a large volume of serum sample in an atmosphere containing physiologic concentrations of blood gases. At the completion of dialysis the dialysate sample for radioimmunoassay quantification can be volumetrically pipetted from the dialysis cell into the RIA tube and the dialysis cell can be discarded.
The following references contain disclosure with respect to dialysis cells: Helenius, T. and Liewendahl, K., "Improved Dialysis Method for Free Thyroxin in Serum Compared with Five Commercial Radioimmunoassays in Nonthyroidal Illness and Subjects with Abnormal Concentrations of Thyroxin-Binding Globulin," Clinical Chemistry, Vol. 29, No. 5, (1983), pages 816-822; Lee, N. D. and Pileggi, V. J., "Measurement of `Free` Thyroxine in Serum," Clinical Chemistry, Vol. 17, No. 3, (1971), pages 166-173; and Elkins, R. P. and Ellis, S. M., "The Radioimmunoassay of Free Thyroid Hormones in Serum" (Excerpta Medica, 7th International Thyroid Conference, Abstract #158 (1976)), pages 597-600; Weeke, J., & Orskov, J., Recent Advances in Clinical Biochemistry (Churchill-Livingston: Edinburgh, N.Y. (1978)), pages 111-128; U.S. Pat. No. 4,077,875 to Kremer issued Mar. 7, 1978.
The dialysis cell described by Helenius et al. has an upper compartment and a lower compartment rather than inner and outer compartments. In assembling that cell any air included in the lower compartment would rise to the membrane and interfere with diffusion of dialyzable substances. The Helenius et al. cell must be assembled with a rubber ring to attach the dialysis membrane to the upper compartment and an aluminum clamping device with two screws to hold the upper and lower compartments tightly together. This cell is not disposable and must be washed and rinsed thoroughly before reuse.
The Ekins et al. cell also consists of an upper compartment and a lower compartment, which raises the problem of trapped air beneath the dialysis membrane that would impede dialysis. In the Ekins et al. cell, the lower compartment is filled with dialysate; then a membrane is stretched across the lower compartment and pressed into the lower compartment by an intermediate unit which contains the serum sample. This in turn is capped by a screw-capped top that closes the entire chamber. This cell is not opened to the environmental air for gas exchange, it is more difficult to assemble, and it is not made of disposable material.
The Lee et al. cell is currently widely used for equilibrium dialysis. It consists of two acrylic plastic halves each of which contains a cut out cavity of matching size as well as holes through which bolts can be placed to attach each half to the other. A dialysis membrane is placed between the halves, the bolts and nuts are tightened (a step which is critical since leaking will cause errors) and the sample is introduced through a narrow port on one side and the buffer through a similar port on the other side. This chamber is expensive and not disposable. It requires considerable effort to wash and prepare the chamber prior to utilization and between assay runs. Furthermore, the ports are too small to allow equilibration with ambient gases or sampling with common quantitative hand-held pipettors.
The dialysis cell illustrated by Weeke et al. consists of dialysis tubing supported in a test tube with a stopper on top of the tube supporting the tubing and closing its two ends. This creates an inner and outer compartment but leaves both blocked from the ambient atmosphere and makes sampling of the inner compartment difficult. Furthermore, the handicraft required to handle wet dialysis tubing, introduce a sample or buffer into the tubing without loss and close both ends after suspending the tubing in the test tube and surrounding it with the test tube contents is a matter of considerable skill.
The Kremer cell is designed to spread the inner compartment contents in a thin layer against the dialysis membrane by filling most of this compartment with solid material. The equipment is non-disposable, and complex to assemble, disassemble, wash and prepare for reuse.